The fallopian tube (FT) is an important reproductive organ in females. The luminal epithelium of the FT is composed of highly polarized secretory and ciliated cells. Recently, accumulating lines of evidence have suggested that the origin of high-grade serous ovarian carcinoma (HGSC) is fallopian tube epithelial cells (FTECs). Due to the lack of a high-fidelity model for FTECs in vitro, homeostasis, differentiation, as well as the transformation of FTECs are still enigmatic. In this study, we optimized the culture condition for the stable expansion of basal stem cells, as well as inducing differentiation of basal cells into polarized secretory and ciliated cells in the air–liquid interface (ALI) condition suitable for long-term culture. This storable culture method of FTECs provides a versatile platform for studying differentiation mechanisms, intercellular communication, and transformation to HGSC, as well as the physiological function of the FT in vitro.